Figure 1. Loss and Reincorporation Rates of GFP:Synapsin I at Individual Synaptic Boutons.

(A) Axons of neurons expressing GFP:Synapsin I. Presynaptic boutons appear as bright puncta along faintly fluorescent axons. The contrast was enhanced nonlinearly in this figure to emphasize axonal fluorescence and demonstrate that the boutons photobleached here did not reside along the same axonal segment. Bar, 10 μm.

(B) Two boutons (arrows) were selectively photobleached by high-intensity laser light, reducing their fluorescence to approximately 30% of their nominal values. Fluorescence recovery at these sites was then followed by time-lapse imaging, initially at 20-s intervals and later at 1-min intervals. GFP:Synapsin I fluorescence levels shown in false color according to color scale near bottom.

(C) At the end of the experiment, presynaptic boutons were labeled (loaded) with FM4–64 by field stimulation (30sec@10hz) followed by unloading (120sec@10hz) to verify the functionality of the photobleached boutons (bottom panels). Note that both photobleached boutons exhibited a capacity for evoked endocytosis and exocytosis of FM4–64. Only boutons that exhibited such a capacity were included in our analysis. Same region as that enclosed in rectangle in (A). Times given in minutes.

(D) Fluorescence recovery time course for photobleached boutons in (A) as well as the mean fluorescence of five nonphotobleached boutons in the same field. Note the gradual reduction of fluorescence in these boutons and its dependence on the sampling rate, indicating that illumination applied during ongoing imaging induces some photobleaching that should be corrected for.

(E) Fluorescence recovery time course after correcting for ongoing photobleaching.

(F) Loss and reincorporation of GFP:Synapsin I molecules at synapses are accelerated by synaptic activity. Neurons were stimulated for 20 s at 20 Hz immediately after collecting the first postbleach images. Despite the brief duration of this stimulation episode, fluorescence recovery was accelerated significantly in comparison to recovery in matched, nonstimulated preparations. Data shown are mean ± standard deviation for all photobleached boutons after normalization as described in Materials and Methods. One-sided error bars only are shown in sake of clarity. The data were fit to a model assuming two pools with different recovery kinetics as described in the text. All experiments were performed in the presence of CNQX (10 μM) and AP-5 (50 μM) to minimize spontaneous activity.